In Situ Imaging of O-Linked β-N-Acetylglucosamine Using On-Tissue Hydrolysis and MALDI Mass Spectrometry

Abstract

Post-translational O-glycosylation of proteins via the addition of N-acetylglucosamine (O-GlcNAc) is a regulator of many aspects of cellular physiology. Processes driven by perturbed dynamics of O-GlcNAcylation modification have been implicated in cancer development. Variability in O-GlcNAcylation is emerging as a metabolic biomarker of many cancers. Here, we evaluate the use of MALDI-mass spectrometry imaging (MSI) to visualize the location of O-GlcNAcylated proteins in tissue sections by mapping GlcNAc that has been released by the enzymatic hydrolysis of glycoproteins using an O-GlcNAc hydrolase. We use this strategy to monitor O-GlcNAc within hepatic VX2 tumor tissue. We show that increased O-GlcNAc is found within both viable tumor and tumor margin regions, implicating GlcNAc in tumor progression.

Keywords: MALDI; O-GlcNAc; O-glycosylation; biomarkers; in situ digestion; mass spectrometry imaging; ultraviolet photodissociation.

Figure 1

Scheme illustrating regulation of O-GlcNAc by nutrients and reciprocal activities of O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA).

Figure 2

MALDI-MS maps showing the composition of five spots after deposition of O-GlcNAc-modified TAB1 glycopeptide on a slide and treatment with OGA. The extracted ion signals correspond to; (A) sodium-cationized glycopeptide (m/z 1760.79), (B) sodium-cationized GlcNAc (m/z 244.08), and (C) sodium-cationized non-glycosylated peptide (m/z 1557.68). The spot on the right side of each panel is a spot of the glycopeptide that had not been treated with OGA. The four spots on the left side of each panel indicate OGA treatment using four different concentrations of OGA, followed by incubation for two hours.

Figure 3

Ion maps of numerous N-glycans were detected by MALDI imaging as sodium-cationized species in different parts of the tissue after PNGase F treatment. (A) m/z 1581.52, Hex7HexNAc2; (B) m/z 1485.49, Hex3dHex1HeNAc4; (C) m/z 1769.06, Hex6dHex1HexNAc3; (D) m/z 1809.59, Hex5dHex1HexNAc4; (E) m/z 2028.19, Hex6HexNAc5; and (F) co-registered collection of glycans. The hematoxylin and eosin (H&E) stain of the same tissue section is shown in Figure 4 for reference.

Figure 4

(A) The H&E stain of a hepatic tumor section contains an outlined tumor region composed of necrotic tumor with viable tumor region found at the interface to the healthy tissue. MALDI imaging of (B) the water-loss oxonium ion of GlcNAc, m/z 204.0865; and (C) the sodium-cationized water-loss oxonium ion, m/z 226.0691. (+) indicates the use of OGA, while (−) indicates an untreated serial section. The tissue sections were treated with PNGase F to remove all N-glycans prior to the OGA (+/−) treatment.

Figure 5

MSI of peptide signatures observed after on-tissue PNGase F and OGA treatments and trypsin digestion (and matched to peptides characterized using in-situ gel trypsin LCMS/MS analysis that correspond to known proteins). (A) Co-registered collection of peptide matches. (B) m/z 1105.60 (−0.9 ppm error) corresponding to vinculin; (C) m/z 2546.27 (−6.5 ppm error) corresponding to fumarate hydratase; (D) m/z 1531.70 (−8.9 ppm error) corresponding to hemoglobin alpha; (E) m/z 1274.70 (−0.1 ppm error) corresponding to myosin heavy chain 9; (F) m/z 1786.87 (−6.1 ppm error) corresponding to ribose-phosphate diphosphokinase; and (G) m/z 2565.26 (6.2 ppm error) corresponding to aminoacyl tryptophan-tRNA synthase. The H&E stain of the same tissue section is shown in Figure S8 for reference and matched peptide sequences and protein identities are reported in Table S3.